BMAP Journal

1. Introduction

Due to slow development over a long period of time with little or no symptoms, sporadic Alzheimer’s disease (AD) is often diagnosed after irreversible neurodegeneration in brains. Thus, early diagnosis of this disease has been a medical priority [1]. This will give opportunities for possible interventions before irreversible neurodegeneration. Two strategies, i.e. special imaging technology of brains and biomarkers, have been under development for early diagnosis [2]. Using biomarkers for early diagnosis obviously has many advantages. It is simple, financially friendly and suitable for large-scale screening. Alzheimer’s disease is \characterized by accumulation of amyloid plaques and neurofibrillary tangles, which are composed of amyloid β peptides and abnormal tau proteins, respectively.

Those abnormal neuronal proteins have been detected in peripheral tissues, the circulation and cerebra-spinal fluid [3-6]. Therefore, numerous studies have explored whether abnormal tau and amyloid peptides in the peripheral tissue or blood could be diagnostic biomarkers [3-7]. Despite many years’ intense studies, it remains unclear about accuracy of those biomarkers in peripheral tissues or blood for early diagnosis [6-9].  IL33 is cytokine, which is widely expressed in astrocytes [10]. We have demonstrated its critical role in regulation of anti-aging or anti-oxidative mechanisms in several organs [11-13]. Importantly, mice lacking IL33 (Il33^-/-) gene develop dementia at old age with similar symptoms to Alzheimer’s [13]. More importantly, those mice also showed accumulation of abnormal tau in their brains [13].

Recently, we further investigated the relationship between tau in peripheral tissues and glymphatic drainage, by comparison of those between WT and Il33^-/- mice.   We have shown that three anti-aging mechanisms, i.e. repair of DNA double-strand-break, autophagy of damaged molecules and glymphatics in neurons or brains in Il33^-/-mice are defected [12,13]. Immunohistochemistry detected PHF in the cortical and hippocampal neurons in Il33^-/- mice after 60 weeks but not in WT mice (Figure 1A). However, we did detect a trace amount of abnormal tau, Paired Helical Fragments (PHF), in brains of WT at 60 weeks by western blot on the cortical tissues (Figure 1B), suggesting generation of abnormal tau in WT mice

A. Western blot detection of PHF in the cortical tissues in various groups of mice as indicated. PHF was quantified by density of PHF bands; n=5. B. Immunohistochemistry reveals accumulation of PHF in cortical neuros (brown). C. Immunofluorescence shows PHF in glomeruli of mice at 60 weeks. D. Summary of PHF in glomeruli based on fluorescent intensity (see C) in mice; n=5.      

Since glymphatic system is able to drain abnormal neuronal proteins and wastes to the circulation, we investigated whether brain PHF tau in WT mice was drained into peripheral tissues. Glomeruli are critical for excretion of body’s wastes. We decided to examine the presence of PHF in kidneys of old WT mice with both immunofluorescence and western blot. Despite a trace amount of abnormal tau in their brains, old WT mice (60 weeks) showed a significant quantity of phosphate tau AT8 and PHF tau in the glomeruli (Figure 1C and D). We next examined abnormal tau in glomeruli of age-matched Il33^-/- mice, which showed a robust accumulation of abnormal tau, i.e. AT8, PHF and insoluble MC1 in their neurons in the cortical and hippocampal regions [13] (Figure 1A)). Unexpectedly, abnormal tau in glomeruli of these Il33^-/- mice was nearly 3 folds lower than WT mice (Figure 1C and D). Thus, quantity of abnormal tau in peripheral tissue or blood did not correlated to their accumulation in the brains, at least in the case of Il33^-/- mice (Figure 1C). Glymphatics play a critical role in drainage of brain wastes [14,15]. We next asked whether deficiency of glymphatics was responsible for the reduction of abnormal tau in peripheral tissues. Aquaporin4 (AQP4) is a critical molecule for generation convective flow to drive wastes to para-venous space of glymphatics [16,17].  We compared AQP4 expression in brains between WT and Il33^-/-mice. RT-PCR revealed a compatible amount of mRNA of brain AQP4 in young Il33^-/- and WT mice (Figure 2A).

A significant increase in brain AQP4 mRNA was then observed in WT mice between 45 and 60 weeks. In contrast, Il33^-/- mice showed a sharp reduction in their brain AQP4 mRNA after 45 weeks, and thus, it led to a 2.5-fold lower of AQP4 mRNA than that in WT at 60 weeks. Although it remains unclear about quantitative relationship between AQP4 and efficacy of glymphatic, a positive correlation between kidney PHF and cortical AQP4 mRNA clearly suggest a dramatic reduction of glymphatic capacity in Il33^-/- mice (Figure 2B). We have previously demonstrated a defect in completion of autophagic digestion in neurons in Il33^-/- mice after 45 weeks (13). Critical role of autophagy in neurons for elimination of neuronal damaged or old proteins has been well known, and its deficiency has been linked to neurodegeneration and Alzheimer’s disease [18-21]. Taking all these together, we hypothesize that quantity of blood/peripheral abnormal tau depends on not only generation rate of abnormal tau, but also their elimination speed by autophagy and drainage by glymphatics. It can be briefly expressed by the following mathematic formula: Tau in neurons, i.e. Tau_neuron = (Tau_generation – Tau_autophgy). Thus, Tau in blood, i.e. Tau_blood = Tau_neuron xglymphatics_efficacy.  This formula may explain why peripheral abnormal tau in WT mice is more thanIl33^-/- mice. Tau_neuroncould be much higher in Il33^-/- mice, which is well demonstrated by accumulation of abnormal tau due to its increased generation and decreased autophagic digestion [13]. However, a robustly smaller coefficient for glymphatics efficacy (i.e. glymphatics_efficacy) in Il33^-/- mice after 45 weeks will be sufficient to bring Tau_blood down to the level, which is much lower than WT mice, despite accumulation of a large quantity of abnormal tau in neurons in thanIl33^-/- mice.  In fact, defective or low glymphatic drainage efficacy has been blamed for tauopathy [15-17].   

2. Conclusion

Abnormal Tau drained to the peripheral tissues or blood has been considered a potential biomarker for diagnosis of dementias such as Alzheimer’s disease. However, abnormal tau concentration in the peripheral tissue or blood was determined not only by its concentration in the diseased brains or generation speed, but also by neuronal autophagy activity and effectiveness of glymphatic drainage. In fact, defective autophagy and glymphatics are critical contribute to Alzheimer’s disease. Thus, it is necessary to identify biomarkers for activity of autophagy and glymphatics in peripheral blood. Only combination of these biomarkers with abnormal tau in peripheral tissues or the circulation may more precisely reflect the status of pathological changes in the brains.

3. Conflict of Interest

All authors declare no conflict of interests.

Figure 1:  Abnormal tau PHF in the cortical tissue and glomeruli in wild type and Il33^-/- mice.

Figure 2: Reduction in cortical AQP4 expression in Il33^-/-mice positively correlates to decreased PHF tau in their glomeruli.  A. Quantitative RT-PCR shows the time course of cortical AQP4 mRNA in mice as indicated. Average quantity of AQP4 mRNA at 20 weeks of WT mice is taken as 1.00 for comparison. B.  Chart shows plot of glomerular PHF versus cortical mRNA for each mouse. Linear regression reveals a positive regression between glomerular PHF and cortical AQP4; also note a low r², suggesting potential involvement of other factors beyond AQP4.        

1. Dubois B, Padovani A, Scheltens P, Rossi A, Dell'Agnello G (2016) Timely Diagnosis for Alzheimer's Disease: A Literature Review on Benefits and Challenges. J Alzheimer’s Dis49: 617-631.
2. Nordberg A (2015) Toward early diagnosis in Alzheimer’s disease. Nat Rev Neurol 11: 69-70.
3. Humpel C (2011) Identifying and validating biomarkers for Alzheimer's disease. Trends in biotechnology29: 26-32.
4. Chen Z, Mengel D, Keshavan A, Rissman RA, Billinton A, et al.  (2018) Learnings about the complexity of extracellular tau aid development of a blood-based screen for Alzheimer's disease, Alzheimer's & Dementia.
5. Sharma N, Singh AN (2016) Exploring Biomarkers for Alzheimer's Disease. J Clin Diagn Res 10: KE01-KE06.
6. Thambisetty M, Lovestone S (2010) Blood-based biomarkers of Alzheimer's disease: challenging but feasible. Biomark Med 4: 65-79.
7. Shi L, Baird AL, Westwood S, Hye A, Dobson R, et al. (2018) A Decade of Blood Biomarkers for Alzheimer's Disease Research: An Evolving Field, Improving Study Designs, and the Challenge of Replication. J Alzheimers Dis    62: 1181-1198.
8. Henriksen K, O'Bryant SE, Hampel H, Trojanowski JQ, Montine TJ, et al. (2014) The future of blood-based biomarkers for Alzheimer's disease. Alzheimer’s & Dementia 10: 115-131.
9. Hampel H, O-Bryan S, Molinuevo JL, Zetterberg H, Masters CL, et al. (2018) Blood-based biomarkers for Alzheimer’s disease: mapping the road to the clinic. Nat Rev Neurol 14: 639-652.
10. Cayrol C, Girard JP (2014) IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. CurrOpin Immunol 31: 31-37.
11. Wu J, Carlock C, Zhou C, Nakae S, Hicks J, et al. (2015) Interleukin33 is required for disposal of unnecessary cells during ovarian atresia through regulation of autophagy and macrophage migration. J Immunol 194: 2140-2147.
12. Carlock C, Wu J, Zhou C, Tatum K, Adams HP, et al. (2014) Unique temporal and spatial expression patterns of IL-33 in ovaries during ovulation and estrous cycle are associated with ovarian tissue homeostasis. J Immunol 193: 161-169.
13. Carlock C, Wu J, Shim J, Moreno-Gonzalez I, Pitcher M, et al. (2017) Interleukin33 deficiency causes tau abnormality and neurodegeneration with Alzheimer-like symptoms in aged mice. Trans Psychiatry 7: e1191.
14. Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, et al. (2015) Structural and functional features of central nervous system lymphatic vessels. Nature 523: 337-341.
15. Tarasoff-Comway JM, Carare R, Osorio RS, Glodzik L, Butler T, et al. (2015) Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol 11: 457-470.
16. Nagelhus E, Ottersen OP (2013) Physiological roles of aquaporin-4 in brain. Physiol Rev93: 1543-1562.
17. Papadopoulos MC, Verkman AS (2013) Aquaporin water channels in the nervous system. Nat Rev Neurosci 14: 265-277.
18. Frake RA, Ricketts T, Menzies FM, Rubinsztein DC(2015) Autophagy and neurodegeneration. J Clin Invest 125: 65-74.
19. Funk KE, Mark RE, Kuret J (2011) Granulovacuolar degeneration (GVD) bodies of Alzheimer's disease (AD) resemble late-stage autophagic organelles. Neuropathol. Appl. Neurobiol37: 295-306.
20. Nixon RA (2013) The role of autophagy in neurodegenerative disease. Nat Med19: 983-997.
21. Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, et al. (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature441: 880-884.
22. This work was supported by NIH R01DK077857 (to YL), NIH R01HD049613 (to YL).